两株识别血吸虫卵相关组分的单克隆抗体靶表位分析

ANALYSIS OF THE TARGET EPITOPES RECOGNIZED BY TWO MONOCLONAL ANTIBODIES DIRECTED TO EGG ASSOCIATED FRACTIONS OF SCHISTOSOMA JAPONICUM

免疫学鉴定两株主要识别卵相关抗原组分的单克隆抗体（２Ｈ１０，２Ｈ１）均属ＩｇＭ同型，能与日本及曼氏血吸虫可溶性卵抗原（ＳＥＡ）及其三氯醋酸可溶组分（ＳＥＡ－ＴＣＡ）形成趋弱阳极或趋中性沉淀带；与虫卵温育均可形成典型环卵沉淀物；两者ＩＦＡ反应谱有明显差异，但均能与肝、肠中的虫卵外围构成明显荧染；经纯化并标记长链生物素后均可建立灵敏度达毫微克水平的同相夹心酶联试验检出相应抗原组分，但２Ｈ１０仅能检出日本血吸虫ＳＥＡ（ＳＥＡｊ），而２Ｈ１则以检出曼氏种ＳＥＡｍ占优。两株单抗的交替异相组合检测，均为阴性，表明两者识别的表位不同。分别经过碘酸钠及三氟醋酸处理ＳＥＡｊ后进行酶联活性测定，显示２Ｈ１０与２Ｈ１所识别的表位均属糖基依赖型，而前者有显著较强的耐氧化性能，进一步佐证了两株单抗识别表位的异源属性。根据理化特性试探了靶组分的液相层析分离，证明２Ｈ１０与２Ｈ１识别的活性基团不为ＣｏｎＡ所有效吸附，应用Ｍｏｎｏ－ＱＦＰＬＣ离子交换层析，活性表位相对集中被０．２—０．４ｍｏｌ／Ｌ离子强度的ＮａＣｌ洗脱峰内。ＳＤＳ－ＰＡＧＥ及免疫印迹试验分析提示了活性组分分子量的不均一性及糖基属性的不可转印特点。

Two IgM isotype monoclonal antibodies(McAb),2H10 and 2H1,recognizing repetitive epitopes on Schistosoma egg associated molecules were characterized and their specificities were identified in a two site sandwich ELISA system.In consistent with the differences in immunological behaviour and specificity demonstrated with immunoelectrophoresis(IEP) and immunofluorescent antibody(IFA) techniques,absolutely negative reactions found in the he terologous detecting system with alternated capture and detecting McAbs of the two revealed a complete incompatibility giving evidences that epitopes on different molecules were recognized.Immunological liability of the target antigen SEA or SEA TCA to the two McAbs were demonstrated on sodium periodate and trifluoroacetic acid treatment indicating the biochemical nature of these epitopes were glycosyolated molecules with apparently higher resistance to the oxidizing agent showing in 2H10 recognizing epitopes. By means of an ion gradient Mono Q FPLC system (Pharmacia),2H10 reactive epitopes of SEA,being tested not so efficiently adsorbed by ConA sepharose affinity column,was found successfully concentrated in the profile eluted with pH 8.0 PBS at 0.2 0.4 NaCl ionic strength. Repeated trials on SDS PAGE and Western blotting analysis with the reactive fractions further showed a heterogeneity of molecular weight range as well as the non transferable property of the CHO reactive groups.